Deoxynojirimycin (DNJ) and certain N-alkylated modifications of this compound are potent endoplasmic reticulum (ER) α-glucosidase I and II inhibitors. (T. D. Butters, et al. Molecular Requirements of Imino Sugars for the Selective Control of N-Linked Glycosylation and Glycosphingolipid Biosynthesis, 11 Tetrahedron: Asymmetry 113-124 (2000).) Imino sugars quickly and efficiently cross the plasma membrane such that the concentration of imino sugars in the cytosol is at equilibrium with the extracellular concentration. (H. R. Mellor, et al., Cellular Effects of Deoxynojirimycin Analogues: Uptake, Retention and Inhibition of Glycosphingolipid Biosynthesis, 381 Biochem. J. 861-866 (2004).)
In the cytosol, imino sugars directly interact with the ceramide-specific glucosyltransferase on the cytosolic side of the cis-Golgi inhibiting glycolipid biosynthesis. However, to modulate N-linked processing by glucosidase inhibition, imino sugars have to gain entry to the ER lumen. The rate of entry into the ER is unknown, but the concentration of imino sugar is assumed to be much lower in the ER lumen than is supplied exogenously to the cell. Evidence for this comes from experiments where the concentration required to inhibit ER glucosidase I has been measured, often requiring 1,000-10,000 times that which inhibits the purified enzyme in vitro. (L. A. van den Broek, et al., Synthesis of Oxygen-Substituted N-alkyl 1-Deoxynojirimycin Derivatives: Aza Sugar α-Glucosidase Inhibitors Showing Antiviral (HIV-1) and Immunosuppressive Activity, 113 Recueil des Travaux Chimiques des Pays-Bas 507-516. (1994).)
Following access to the lumen of the ER, DNJ analogues inhibit the removal of glucose residues, mediated by α-glucosidases I and II, forming proteins containing hyperglucosylated N-linked oligosaccharides that may fail to interact with the chaperones calnexin and calreticulin, both of which are involved in protein folding quality control. (R. G. Spiro, et al., Definition of the Lectin-like Properties of the Molecular Chaperone, Calreticulin, and Demonstration of Its Copurification with Endomannosidase from Rat Liver Golgi, 271 J. Biol. Chem. 11588-11594 (1996).) Some proteins with hyperglucosylated glycans may still be processed in the Golgi by an endo-α(1,2)mannosidase, thus circumventing the block in oligosaccharide processing caused by glucosidase inhibition. (K. Fujimoto, K., et al., α-Glucosidase II-deficient Cells Use Endo α-Mannosidase as a Bypass Route for N-Linked Oligosaccharide Processing, 266 J. Biol. Chem. 3571-3578 (1991); S. E. Moore, et al., Demonstration That Golgi Endo-α-D-mannosidase Provides a Glucosidase-independent Pathway for the Formation of Complex N-Linked Oligosaccharides of Glycoproteins, 265 J. Biol. Chem. 13104-13112 (1990).)
The removal of misfolded protein from the ER and production of free oligosaccharides (FOS) is a normal cellular process. Calnexin- or calreticulin-dependent, aberrantly-folded protein and hyperglucosylated, aberrantly-folded proteins are ultimately translocated out of the ER into the cytosol via the Sec61p channel (E. J. Wiertz, et al., Sec61-mediated Transfer of a Membrane Protein from the Endoplasmic Reticulum to the Proteasome for Destruction, 384 Nature 432-438 (1996)), where the N-linked oligosaccharide is released by a cytosolic peptide: N-glycanase (PNGase) (which may or may not be in direct interaction with the Sec61p channel) producing FOS. (G. Li, et al., Multiple Modes of Interaction of the Deglycosylation Enzyme, Mouse Peptide N-glycanase, with the Proteasome, 102 Proc. Natl. Acad. Sci. USA 15809-15814 (2005); Spiro, R. G., Role of N-linked Polymannose Oligosaccharides in Targeting Glycoproteins for Endoplasmic Reticulum-associated Degradation, 61 Cell Mol. Life Sci. 1025-1041 (2004).) This process of selective protein export from the ER to the cytosol followed by proteasomal degradation is known as ER-associated degradation (ERAD). FOS produced in the cytoplasm are acted upon by cytosolic enzymes such as endo-R-Nacetylglucosaminidase (EnGNase) (T. Suzuki, et al., Endo-β-N-acetylglucosaminidase, an Enzyme Involved in Processing of Free Oligosaccharides in the Cytosol, 99 Proc. Natl. Acad. Sci. USA 9691-9696 (2002)) and cytosolic a-mannosidase (V. A. Shoup, et al., Purification and Characterization of the a-D-Mannosidase of Rat Liver Cytosol, 251 J. Biol. Chem. 3845-3852 (1976)), ultimately forming a Man5GlcNAc1 (M5N) species that is transported to the lysosome. However, glucosylated FOS are allegedly not able to gain entry to the lysosome for degradation (A. Saint-Pol, et al., Cytosol-to-lysosome Transport of Free Polymannose-type Oligosaccharides, 274 J. Biol. Chem. 13547-13555 (1999)), and their fate remains to be determined. Other small, but detectable, amounts of FOS including Glc1Man5GlcNAc1 are present in cells, in addition to M5N, representing the normal default pathway for ERAD. (H. R. Mellor et al., Cellular Effects of Deoxynojirimycin Analogues: Inhibition of N-Linked Oligosaccharide Processing and Generation of Free Glucosylated Oligosaccharides, 381 Biochem. J. 867-875 (2004).)
The development of a cellular-based ER α-glucosidase assay that determines the rate of α-glucosidase-mediated hydrolysis of N-linked oligosaccharides, as proteins are folded in the ER in the presence of inhibitor, reveals important principles of oligosaccharide intermediates in the biosynthetic pathway and can be used to predict efficacy for protein misfolding; a strategy that has been proposed as a potential therapy for the inhibition of viral infectivity. (R. A. Dwek, et al., Targeting Glycosylation as a Therapeutic Approach, 1 Nat. Rev. Drug Discov. 65-75 (2002).)